1. Field of the Invention
The present invention relates generally to immunoassays, receptor-, cellular-, and molecular-based assays, and liquid delivery devices incorporating the same. More specifically it relates to an analytical assay or test device containing a liquid delivery element and may contain reagents for detection of an analyte of interest.
2. Discussion of Related Art
Various chromatographic and microfluidic immunoassay techniques have been available for some time. For example, immune-based latex agglutination tests for detecting a factor associated with rheumatoid arthritis were used as early as 1956 (Singer et al., Am. J. Med. 22:888-892 (1956)). Tests that can be performed with such chromatographic and fluid systems often involve immunoassays, which depend on the specific interaction between an antigen and a corresponding antibody. Immunoassays therefore have gained consideration as an important and convenient means of testing for the presence or the amount, or both, of clinically important molecules.
Among the many analytical systems used for detection of analytes, particularly those of biological interest, are chromatographic and fluidic assay systems. Among the analytes frequently assayed with such systems are: (1) hormones, such as human chorionic gonadotropin (hCG), which is frequently assayed as a marker of human pregnancy; (2) antigens, particularly antigens specific to bacterial, viral, and protozoan pathogens, such as streptococcus, hepatitis virus, and giardia; (3) antibodies, particularly antibodies induced as a result of infection with pathogens, such as bacteria or viruses, such as HIV; (4) other proteins, such as hemoglobin, frequently assayed in determinations of fecal occult blood, an early indicator of gastrointestinal disorders such as colon cancer; (5) enzymes, such as aspartate aminotransferase, lactate dehydrogenase, alkaline phosphatase, and glutamate dehydrogenase, frequently assayed as indicators of physiological function and tissue damage; (6) drugs, both therapeutic drugs, such as antibiotics, tranquilizers, and anticonvulsants, and illegal drugs of abuse, such as cocaine, heroin, and marijuana; (7) vitamins; and (8) nucleic acid material.
Such chromatographic systems are frequently used by physicians and medical technicians for rapid in-office diagnosis. They are therefore commonly referred to as “point of care” (POC) devices. These tests may also be used for therapeutic monitoring of a variety of conditions and disorders. They are also increasingly used by: patients themselves for at-home monitoring of such conditions and disorders; scientists for use in field testing for transgenic crops and environmental contaminates; soldiers in battlefield conditions for biological warfare weapon detection; and veterinary and emergency technicians where rapid testing is crucial.
The chromatographic and fluidic techniques used in conjunction with most common immunoassays involve the principle of immunochromatography. In general, this technique uses a label or indicator particle that has been linked to an immunoprotein specific for the molecule to be assayed. The label and antibody/antigen together are referred to as a conjugate, which is then mixed with a specimen. If the analyte molecule is present in the specimen, the conjugate specifically binds to the molecule. The label aspect provides a detectable indication that the molecule to be assayed is present. The specific reactions that are employed vary with the nature of the molecule being assayed and the sample to be tested. Such determinations are readily made depending on the molecule of interest.
Immunochromatographic and fluidic assays fall into two principal categories: “sandwich” and “competitive,” according to the nature of the antigen-antibody complex to be detected and the sequence of reactions required to produce that complex. In the case of antigen detection, the sandwich immunochromatographic procedures call for mixing the sample that may contain the analyte to be assayed with antibodies to the analyte. These antibodies are mobile and typically are linked to a label or a reagent, such as dyed latex, a colloidal metal sol, or a radioisotope. This mixture is then applied to a chromatographic medium containing a band or capture zone. This band or capture zone contains immobilized antibodies for the analyte of interest. The chromatographic medium can also be in the form of a strip resembling a dipstick. When the complex of the molecule to be assayed and the labeled antibody reaches the zone of the immobilized antibodies on the chromatographic medium, binding occurs, and the bound-labeled antibodies are localized at the zone. This indicates the presence of the molecule to be assayed. This technique can be used to obtain qualitative results. Examples of sandwich immunoassays performed on test strips are described in U.S. Pat. Nos. 4,168,146 to Grubb et al., 4,366,241 to Tom et al., 6,017,767 and 5,998,220 to Chandler; and 4,305,924 to Piasio et al.
In competitive or indirect immunoassays, the immobilized component is typically present in controlled amounts and the mobile component is present in unknown amounts. The unknown amount of mobile component is supplemented with a known amount of the same component that has been tagged by the addition of a measurable constituent which does not interfere with its immunochemical reactive properties. The tag may consist of a radioisotope, a chromophore, a particle, a fluorophor, or an enzyme. The amount of tagged material bound immunochemically to the solid phase will depend upon the amount of untagged component in solution competing for the same binding sites. The more of the unknown component present, the less will be the amount of bound tagged component. As such a relative determination can be made.
Enzyme-based chromatographic assays have gained use in addition to immunochromatographic assays. These enzyme-based assays involve an enzymatically-catalyzed reaction instead of an antigen-antibody reaction. The enzymatically-catalyzed reaction frequently generates a detectable product.
Although useful, currently available chromatographic techniques using test strips have a number of drawbacks. Some samples, for example, fecal samples, contain particulate matter that can obscure or color the pores of the chromatographic medium, greatly hindering detection of the labeling reagents. Blood for example, obviously contain cells and colored components that obscure the color generation in the test, and therefore make it difficult, if not impossible, to read. Blood cells also tend to clog the pores in the medium. Wet chromatographic medium is also sometimes difficult to read because of specular reflection from the chromatography medium. There are various other drawbacks to chromatographic techniques, including physical properties of lateral flow, fluid front movement along the strip, and color generation intensity and location.
Sample preparation and waste generation are responsible for other problems with currently available devices and techniques for fluidics and immunochromatography. The increased prevalence of diseases spread by infected blood and blood fractions, such as HIV and hepatitis, has only exacerbated these concerns. The available forms of lateral flow devices have a large portion of their components that are only used for mechanical support of the chromatographic membrane, and are not sealed. Therefore disposal is a concern, expensive, and possibly hazardous because of the presumed bio-hazards. Precautions have to be taken so that workers, or people who may inadvertently come into contact with the waste, do not themselves become contaminated.
One common aspect of known devices, particularly in lateral flow technology and microfluidic systems, is that the assay is read visually, that is, by means of one or more optically readable lines on a test strip held in a carrier or through “windows” in the device, which may have various configurations. As briefly indicated above, there are several limitations or disadvantages to the known optically detected assays. Because they are optical, only surface changes (typically coloration) can be detected. In addition, these tests are only appropriate where the sample solution is colorless. Also, the target analytes may be in the sample solution but of such a low concentration that only relatively few are captured in the capture zone of the assay. This may provide a faint or even non-optically detectable reading, and a false negative reading can result. Quantitative assessments are only an estimation based on color intensity of the detection line. Because the prior art assays are optically read, they are subject to contamination by exposure and light-caused degradation. Therefore, they have a limited archival shelf life.
Typically one end of the test is exposed to the sample, normally a fluid of some type, being tested for the particular target analytes of interest. The fluid migrates through a capillary or chromatographic medium whereby the analyte with its label is captured and immobilized, while the remaining fluid is absorbed into a medium at the distal end of the assay. Examples of optically read lateral flow devices and methods are shown in U.S. Pat. Nos. 5,591,645; 5,798,273; 5,622,871; 5,602,040; 5,714,389; 5,879,951; 4,632,901; and 5,958,790.
Many current devices also have a liquid sample application member in direct fluid communication with the test strip. Typically this member is made from an absorbent material that may be contained within the device itself, or may protrude from the device to be more easily introduced to the liquid sample. The absorbent liquid sample application member attempts to control the rate of flow of fluid through the device. The concern is that if the liquid sample is applied directly to the test strip, the strip may be easily flooded and the assay rendered ineffective. Also the application member is usually made from a different material than the test strip itself due to the relatively large quantity of liquid that it is expected to manage.
Others have attempted to control the rate of fluid flow to the test strip by employing capillary assay formats. Examples of capillary assays can be found in U.S. Pat. Nos. 4,883,760 and 5,474,902. However, these are not an appropriate scale for use in point-of-care situations.
Biological systems other than lateral flow immunoassays have employed magnetic particles or microbeads, which may be more specifically referred to as superparamagnetic iron oxide impregnated polymer beads. These beads bind with the target analytes in the sample being tested and are then typically isolated or separated out magnetically. Once isolation has occurred, further testing may be conducted, including observing particular images, whether directly optically or by means of a camera. Examples of these systems may be found in U.S. Pat. Nos. 3,981,776; 5,395,498; 5,476,796; 5,817,526; and 5,922,284.
Another apparatus for detecting target molecules in a liquid phase is shown in U.S. Pat. No. 5,981,297 where magnetizable particles are employed and the output of magnetic field sensors indicates the presence and concentration of target molecules in the sample being tested. Other examples to sense magnetically using physical forces are disclosed in U.S. Pat. Nos. 5,445,970; 5,981,297; and 5,925,573. However, in these devices, the magnet requires relatively high power because the gap where the assay is placed must be wide enough to accommodate the relatively thick assay device.
Accordingly, it would be advantageous to have a testing device where the fluid sample is applied in such a manner that avoids the problems of prior art devices, that has a detection region providing standardized, reliable, and reproducible results, and that is also archival for storage over time. The present invention satisfies these needs and provides related advantages as well.